A new method to reveal the genotoxic effects of N-nitrosodimethylamine in pregnant mice

DNA damage and repair in kidney and liver of mouse fetuses exposed to selected doses of N-nitrosodimethylamine (NDMA) (CAS No. 62.75.9) were studied using the alkaline elution technique. CD1 female mice (15 days pregnant) were treated i.p. with 2 and 10 mg/kg b.w. of NDMA; a slight increase in DNA damage was observed in their fetuses compared to untreated controls. A 2-fold higher extent of DNA damage was induced when mice were treated by intrafetal injections of a rat S9 activating fraction (S9) immediately before exposure to the same dose of NDMA by transplacental means. The DNA-strand breaks disappeared as a function of time in animals treated with NDMA alone. In contrast, a significant persistence of DNA damage was detected in the liver and lung of fetuses which were treated with S9 and NDMA in sequence. These experiments demonstrate the metabolic immaturity of unborn mice as far as the carcinogenic activation of NDMA is concerned and show the high susceptibility of fetal tissues to DNA-damaging agents. The alkaline elution applied in vivo by the transplacental route combined with the intrafetal injection of an exogenous activating microsomal fraction allow to extend our knowledge on the interaction of metabolism-dependent chemicals with fetal tissues.     

广西HBsAg阳性母亲的胎儿肝、肾细胞中乙肝病毒DNA存在状态的研究

采用核酸分子杂交Ｓｏｕｔｈｅｒｎ印迹法,以３２Ｐ标记的ＨＢＶＤＮＡ为探针,检测ＨＢｓＡｇ阳性母亲引产的４０例胎儿的肝、肾组织。结果有２例胎肝和１例胎肾细胞ＤＮＡ出现大于３．２ｋｂ的杂交带,表明ＨＢＶＤＮＡ已处于整合状态。胎肾细胞基因组中查出ＨＢＶＤＮＡ整合为首次报道。     

DNA damage in tissues and organs of mice treated with diphenyl diselenide

Diphenyl diselenide (DPDS) is an organoselenium compound with interesting pharmacological activities and various toxic effects. In previous reports, we demonstrated the pro-oxidant action and the mutagenic properties of this molecule in bacteria, yeast and cultured mammalian cells. This study investigated the genotoxic effects of DPDS in multiple organs (brain, kidney, liver, spleen, testes and urinary bladder) and tissues (bone marrow, lymphocytes) of mice using in vivo comet assay, in order to determine the threshold of dose at which it has beneficial or toxic effects. We assessed the mechanism underlying the genotoxicity through the measurement of GSH content and thiobarbituric acid reactive species, two oxidative stress biomarkers. Male CF-1 mice were given 0.2-200 micromol/kg BW DPDS intraperitonially. DPDS induced DNA damage in brain, liver, kidney and testes in a dose response manner, in a broad dose range at 75-200 micromol/kg with the brain showing the highest level of damage. Overall, our analysis demonstrated a high correlation among decreased levels of GSH content and an increase in lipid peroxidation and DNA damage. This finding establishes an interrelationship between pro-oxidant and genotoxic effects. In addition, DPDS was not genotoxic and did not increase lipid peroxidation levels in any organs at doses < 50 micromol/kg. Finally, pre-treatment with N-acetyl-cysteine completely prevented DPDS-induced oxidative damage by the maintenance of cellular GSH levels, reinforcing the positive relationship of DPDS-induced GSH depletion and DNA damage. In summary, DPDS induces systemic genotoxicity in mammals as it causes DNA damage in vital organs like brain, liver, kidney and testes.     

Effect of prolactin on DNA methylation in the liver and kidney of rat

Prolactin is an important growth modulatory hormone in fetal and adult tissues. It stimulates DNA synthesis and enzymatic markers of the G1 phase of cell cycle in rat liver and other tissues. In this study the effects of prolactin on 5-methyl cytosine content in liver and kidney of rats was studied using HPLC. Prolactin treatment caused hypomethylation of DNA in the liver and kidney of immature rats at 48 h after treatment and the effect remained even at 72 h. Prolactin also caused hypomethylation of DNA in the kidney and liver of adult rats at 48 h after treatment. These results indicate that prolactin probably regulates DNA methylation in the liver and kidney of immature and adult rats.     

Organ-specific DNA damage induced in mice by the organotropic carcinogens 4-nitroquinoline 1-oxide and dimethylnitrosamine.

The extent of DNA fragmentation induced in lung, kidney, and liver of mice injected with the chemical carcinogens 4-nitroquinoline 1-oxide (4NQO), dimethylnitrosamine (DMN) and the noncarcinogenic 4-aminoquinoline 1-oxide (4AQO) was estimated by the alkaline sucrose gradient technique. A floating of minced lung tissue pieces in the alkaline lysing solution on top of the gradients afforded a gentle method of lung DNA extraction. This technique minimized mechanical shearing of lung DNA and permitted comparisons to be made with liver and kidney DNA sedimentation patterns. The extent of DNA damage induced by 4NQO followed the order: lung, kidney, liver, while that induced by DMN followed the order: liver, kidney, lung. The sites of greatest DNA damage appeared to correlate with sites of high levels of DNA repair synthesis and the sites of tumor induction. No DNA damage was induced by the noncarcinogenic 4-aminoquinoline 1-oxide (4AQO).     

DNaseI sensitivity of the rat albumin and alpha-fetoprotein genes.

We have analyzed the DNaseI sensitivity of chromatin from the rat albumin and alpha-fetoprotein genes in the fetal liver (which synthesizes albumin and alpha-fetoprotein), adult liver (which synthesizes albumin), fetal yolk sac (which synthesizes alpha-fetoprotein), and adult kidney (which synthesizes neither). Active genes were much more sensitive than their kidney counterparts, and the adult liver alpha-fetoprotein and fetal yolk sac albumin genes showed intermediate levels of sensitivity. Sensitivity was analyzed as a function of the extent of DNaseI digestion. Rate constants were calculated for the degradation of individual DNA hybridization bands and normalized to the intrinsic rate constants of the same bands degraded in purified DNA. This enabled us to eliminate the inconsistencies that otherwise result from comparing chromatin sensitivity of different DNA sequences, or chromatin sensitivity in different nuclear environments.     

Formation and subsequent removal of O6-methylguanine from deoxyribonucleic acid in rat liver and kidney after small doses of dimethylnitrosamine

1. The amounts of 7-methylguanine and O⁶-methylguanine present in the DNA of liver and kidney of rats 4h and 24h after administration of low doses of dimethylnitrosamine were measured. 2. O⁶-Methylguanine was rapidly removed from liver DNA so that less than 15% of the expected amount (on the basis of 7-methylguanine found) was present within 4h after doses of 0.25mg/kg body wt. or less. Within 24h of administration of dimethylnitrosamine at doses of 1mg/kg or below, more than 85% of the expected amount of O⁶-methylguanine was removed. Removal was most efficient (defined in terms of the percentage of the O⁶-methylguanine formed that was subsequently lost within 24h) after doses of 0.25–0.5mg/kg body wt. At doses greater or less than this the removal was less efficient, even though the absolute amount of O⁶-methylguanine lost during 24h increased with the dose of dimethylnitrosamine over the entire range of doses from 0.001 to 20mg/kg body wt. 3. Alkylation of kidney DNA after intraperitoneal injections of 1–50μg of dimethylnitrosamine/kg body wt. occurred at about one-tenth the extent of alkylation of liver DNA. Removal of O⁶-methylguanine from the DNA also took place in the kidney, but was slower than in the liver. 4. After oral administration of these doses of dimethylnitrosamine, the alkylation of kidney DNA was much less than after intraperitoneal administration and represented only 1–2% of that found in the liver. 5. Alkylation of liver and kidney DNA was readily detectable when measured 24h after the final injection in rats that received daily injections of 1μg of [³H]dimethylnitrosamine/kg for 2 or 3 weeks. After 3 weeks, O⁶-methylguanine contents in the liver DNA were about 1% of the 7-methylguanine contents. The amount of 7-methylguanine in the liver DNA was 10 times that in the kidney DNA, but liver O⁶-methylguanine contents were only twice those in the kidney. 6. Extracts able to catalyse the removal of O⁶-methylguanine from alkylated DNA in vitro were isolated from liver and kidney. These extracts did not lead to the loss of 7-methylguanine from DNA. 7. The possible relevance of the formation and removal of O⁶-methylguanine in DNA to the risk of tumour induction by exposure to low concentrations of dimethylnitrosamine is discussed.     

Organ-specific DNA damage of tris(2,3-dibromopropyl)-phosphate and its diester metabolite in the rat.

The organ specificity of tris(2,3-dibromopropyl)phosphate(Tris-BP)-induced DNA damage was investigated in the rat 2 h after a single i.p. injection of 350 mumol/kg. Extensive DNA damage, measured with the alkaline elution method, was found in the kidney, liver and small intestine. Less, but significant DNA damage was detected in the brain, lung, spleen, large intestine and testis. The role of different pathways in the activation of Tris-BP to DNA damaging products was studied in isolated liver and testicular cells. Concentrations as low as 2.5-5 microM Tris-BP caused DNA damage in the hepatocytes, whereas an approximately 10-fold higher concentration was needed in testicular cells to produce a similar amount of DNA damage. Depletion of GSH by diethyl maleate (DEM) did not affect the extent of DNA damage caused by Tris-BP in the liver cells, but blocked the genotoxic effect in testicular cells. Two specifically deuterated Tris-BP analogs, C3D2-Tris-BP and C2D1-Tris-BP, were significantly less potent in causing DNA damage than the protio compound in isolated liver cells and were somewhat less potent in testicular cells. The major urinary metabolite of Tris-BP, bis(2,3-dibromopropyl)phosphate (Bis-BP), was less potent than Tris-BP in causing kidney DNA damage after in vivo exposure. Furthermore, Bis-BP induced substantially less DNA damage in isolated liver and testicular cells. Similar to the effect of DEM on the DNA damage caused by Tris-BP, the DNA damage caused by Bis-BP could be decreased by DEM-pretreatment in testicular cells but not in liver cells. The present study shows that Tris-BP is a potent multiorgan genotoxic agent in vivo. The in vitro data indicate that P-450 mediated metabolism of Tris-BP is more important than activation by glutathione S-transferases of Tris-BP in liver cells, whereas the latter activation pathway seems to be most important in testicular cells.     

The absorption and metabolism in rats of small oral doses of dimethylnitrosamine. Implication for the possible hazard of dimethylnitrosamine in human food.

1. Groups of rats were given one dose of the carcinogen dimethylnitrosamine by gastric intubation. The dose was varied between 10mg/kg body wt. and 1 microgram/kg body wt. 2. The dose was rapidly absorbed. 3. The methylation of liver DNA resulting from the administration of this carcinogen was proportional to dose. This suggests that small doses are absorbed from the gut with no more loss than large doses. 4. As the dose was decreased there was a disproportionately greater decrease in the alkylation of kidney DNA, and when the dose was less than 40 microgram/kg body wt. the methylation of kidney DNA was no longer detectable. This possibly explains why small amounts of dimethylnitrosamine in the diet do not induce kidney tumours. 5. Comparison of the relative alkylation of liver DNA and kidney DNA resulting from an oral and from an intravenous dose of dimethylnitrosamine suggest that small amounts of dimethylnitrosamine absorbed into the portal blood from the gut are completely metabolized by the liver and do not enter the general circulation. 6. The implications of these results for the possible hazard of dimethylnitrosamine in human food is discussed.     

Gamma ray induced genetic changes in different organs of chick embryo using peripheral blood micronucleus test and comet assay

Ionizing radiation is known to produce a variety of cellular and sub cellular damage in both prokaryotic and eukaryotic cells. Present studies were undertaken to assess gamma ray induced DNA damage in different organs of the chick embryo using alkaline comet assay and peripheral blood micronucleus test. Further the suitability of chick embryo, as an alternative model for genotoxicity evaluation of environmental agents was assessed. Fertilized eggs of Rhode island red strain were exposed to 0.5, 1 and 2 Gy of gamma rays delivered at a dose rate of 0.316 Gy/min using a ⁶⁰Co teletherapy machine. Peripheral blood smears were prepared from 8- to 11-day-old chick embryos for micronucleus test. Alkaline comet assay was performed on 11-day-old chick embryos in different organs such as the heart, liver, lung, blood, bone marrow, brain and kidney.Analysis of the data revealed a significant increase in the frequency of micronucleated polychromatic erythrocytes, micronucleated normochromatic erythrocytes and total micronucleated erythrocytes in the peripheral blood of gamma irradiated chick embryos at all the doses tested as compared to the respective controls. The polychromatic to normochromatic erythrocytes ratio which is an indicator of proliferation rate of hematopoetic tissue, decreased in the irradiated groups as compared to the controls. Data obtained from comet assay, clearly demonstrated a significant increase in DNA strand breaks in all the organs of irradiated chick embryos as compared to the respective controls. However, maximum damage was observed in the heart tissue on all the doses tested, followed by kidney, brain, lung, blood and liver. The lowest damage was observed in the bone marrow tissue. Both micronucleus test and comet assay were found to be suitable biomarkers for the evaluation of genotoxicity of gamma radiation in the chick embryo.     

Alkaline, Endo III and FPG modified comet assay as biomarkers for the detection of oxidative DNA damage in rats with experimentally induced diabetes

Increased production of reactive oxygen species under diabetic condition underlines the higher oxidatively damaged DNA in different tissues. However, it is practically difficult to assess the oxidatively damaged DNA in different internal organs. Therefore, the present study was aimed to evaluate the extent of oxidative stress-induced DNA damage in different organs with the progression of diabetes. Diabetic and control Sprague Dawley rats were sacrificed in time-dependent manner and the lung, liver, heart, aorta, kidney, pancreas and peripheral blood lymphocytes (PBL) were analyzed for both alkaline and modified comet assay with endonuclease-III (Endo III) and formamidopyrimidine-DNA glycosylase (FPG) (hereafter called modified comet assay) for the detection of oxidative DNA damage. The statistically significant increase in olive tail moment (OTM) was found in all the tested tissues. The extent of DNA damage was increased with the progression of diabetes as revealed by the parameter of OTM in alkaline and modified comet assay. Further, the positive correlations were observed between OTM of the lung, liver, heart, aorta, kidney and pancreas with PBL of diabetic rat in the alkaline and modified comet assay. Moreover, significant increase in the 8-oxodG positive nuclei in the lung, liver, heart, aorta, kidney and pancreas was observed in 4th and 8th week diabetic rat as compared to control. Results of the present study clearly indicated the suitability of alkaline and modified comet assay for the detection of multi-organ oxidative DNA damage in streptozotocin (STZ)-induced diabetic rat and showed that damaged DNA of PBL can be used as a suitable biomarker to assess the internal organs response to DNA damage in diabetes.     

Cadmium-induced oxidative stress and DNA damage in kidney of pregnant female rats

In the present study, we have investigated the influence of sub-acute treatment with cadmium (Cd) on some parameters indicative of oxidative stress and DNA damage in tissues of pregnant female rats. Pregnant female rats (n=6) were injected subcutaneously, daily with a dose of cadmium chloride of 3 mg/kg body weight (b.w.) from day 6 to day 19 of pregnancy, and they were allowed to deliver normally. MDA level and GPx, CAT and SOD activities were used as markers of oxidative stress in liver and kidney. The 8-oxo-dG level was measured by the HPLC-EC system. Cd treatment increased MDA (+116%, p<0.01) in kidney. Moreover, Cd treatment also decreased CuZn-SOD (-11%, p<0.05) and GSH level (-52%, p<0.05) in kidney. Treated rats displayed an increase of the liver metallothionein (MT) level. Induction of MT in liver was probably implicated in the detoxification of Cd. The high level of Cd (3 mg/kg) used in the present study is partially neutralized by MT in liver, whereas the free fraction could be implicated in the oxidative stress and DNA oxidation observed in kidney. Cd treatment failed to alter 8-oxodGuo, indicating the absence of DNA oxidation in liver; by contrast, the same treatment increased the 8-oxodGuo level (+51%, p<0.05) in the kidney of pregnant female rats, indicating an oxidative stress associated with DNA damage only in kidney.     

Exposure to bisphenol A during embryonic/fetal life and infancy increases oxidative injury and causes underdevelopment of the brain and testis in mice

We investigated the modifications in endogenous antioxidant capacity and oxidative damage in the brain, liver, kidney and testis in mice exposed to bisphenol A (BPA), an environmental endocrine disrupter. Mice were exposed to BPA throughout embryonic/fetal life and during lactation by feeding their pregnant/lactating mothers BPA at 5 or 10 microg per milliliter of drinking water. At the age of four weeks, male mice were sacrificed. Exposure to BPA increased the activity of catalase and glutathione peroxidase in the liver and kidney, respectively. It also increased thiobarbituric acid-reactive substances in the brain, kidney and testis, and decreased the wet weight of the brain, kidney and testis. Our results suggest that exposure to BPA throughout embryonic/fetal life and during infancy induces tissue oxidative stress and peroxidation, ultimately leading to underdevelopment of the brain, kidney and testis.     

Response of selected fetal organs to antineoplastic agents

This study was designed to quantitate the effects of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DIC) and 5-(3,3-bis(2-chlorethyl)-1-triazeno)-imidazole-4-carboxamide (BIC) on growth and selected components of rat fetal organs. Twelve-day pregnant rats were given single intraperitoneal injections of 600 mg/kg of DIC and 900 mg/kg of BIC and autopsied on day 21 of gestation. Fetal liver, brain, kidney, and placenta were removed, weighed, and assayed for total DNA, RNA, and protein. DIC significantly reduced weight, total DNA, RNA, and protein of all four fetal organs as compared to age-matched controls. The brain was most severely affected by this compound. BIC also significantly reduced weight, DNA, RNA, and protein of fetal brain, kidney, and placenta, but in fetal liver only weight and total protein were significantly depressed, while DNA and RNA remained essentially unchanged. The effect of BIC was maximal on the placenta.     

Organ and gestational age effects of maternal nutrient restriction on global methylation in fetal baboons

Background  A sub-optimal intrauterine environment alters the trajectory of fetal development with profound effects on life-time health. Altered methylation, a proposed epigenetic mechanism responsible for these changes, has been studied in non-primate species but not nonhuman primates. We tested the hypotheses that global methylation in fetal baboon demonstrates organ specificity, gestational age specificity, and changes with maternal nutritional status.
Methods  We measured global DNA methylation in fetuses of control fed (CTR) and nutrient restricted mothers fed 70% of controls (MNR) for brain, kidney, liver and heart at 0.5 and 0.9 gestation (G).
Results  We observed organ and gestation specific changes that were modified by maternal diet. Methylation in CTR fetuses was highest in frontal cortex and lowest in liver. MNR decreased methylation in 0.5G kidney and increased methylation in 0.9G kidney and frontal cortex.
Conclusion  These results demonstrate a potential epigenetic mechanism whereby reduced maternal nutrition has long-term programming effects on fetal organ development.     

花色苷提取物对大鼠发育关键期铅致肝脏和肾脏损伤的保护作用研究

为研究天然色素花色苷（anthocyanins,ACNs）对铅中毒引起的脏器损伤的保护作用,构建了铅暴露大鼠模型,灌服不同剂量的ACNs溶液和强力排铅药二巯基丁二酸（dithioglysuccinic acid, DMSA）,连续3周,于末次给药24 h后考察脏器中的铅含量、组织病理学、血液及脏器生化指标;并采用综合生物标志物响应（integrated biomarker responses, IBR）评估ACNs对铅致发育期大鼠肝脏和肾脏氧化损伤的修复能力。实验结果表明,铅大量存在于大鼠的肝脏和肾脏组织中,造成脏器病理学结构及氧化损伤;而ACNs可促进铅排出机体,改善组织病理学结构,使血清中转氨酶、肌酐等生化指标水平明显好转,并升高肝和肾抗氧化酶的活性,减少还原性物质的含量,改善铅诱导组织的氧化损伤;IBR分析结果显示,ACNs可使铅损伤的脏器得到明显的修复。结果表明,ACNs营养干预可有效拮抗铅致机体的氧化损伤,从而有效修复铅损伤的肝肾组织。     

Lipid peroxidation in mitochondria and microsomes from adult and fetal rat tissues

Pregnant female Wistar rats that received a control (100 ppm Zn) or a Zn-deficient diet (1.5 ppm Zn) from d 0 to 21, or nonpregnant normally fed female rats without or with five daily oral doses of 300 mg/kg salicylic acid were used for the experiments. In isolated mitochondria or microsomes from various maternal and fetal tissues, lipid peroxidation was determined as malondialdehyde formation measured by means of the thiobarbiturate method. Zn deficiency increased lipid peroxidation in mitochondria and microsomes from maternal and fetal liver, maternal kidney, maternal lung microsomes, and fetal lung mitochondria. Lipid peroxidation in fetal microsomes was very low. Zn deficiency produced a further reduction of lipid peroxidation in fetal liver microsomes. Salicylate increased lipid peroxidation in liver mitochondria and microsomes after addition in vitro and after application in vivo. The increase of lipid peroxidation by salicylate may be caused by two mechanisms: an increased cellular Fe uptake that, in turn, can increase lipid peroxidation and chelating Fe, in analogy to the effect of ADP in lipid peroxidation. The latter effect of salicylate is particularly expressed at increased Fe content.     

Ontogeny of ovine fetal liver and kidney plasma membrane insulin receptors and fetal growth

This investigation was performed to define certain characteristics of insulin-receptor interaction during the last 2 months of gestation in fetal sheep liver and kidney. Twenty-one sheep carrying a total of 46 fetuses were sacrificed at various gestational ages from 94 days to term; fetal and maternal livers and kidneys were analyzed by a radioreceptor assay for insulin binding characteristics. Specific binding of insulin to partially purified ovine fetal liver and kidney plasma membranes increased as gestation approached term, at which time specific binding was two- to fourfold greater to fetal than to maternal tissues. Associated with increased specific binding were late gestational increases in affinity of insulin for receptors in both fetal liver and kidney and an earlier increase in insulin receptor concentration in fetal kidney. These observations in fetal sheep liver and kidney are similar to reported observations in other species. However, the increase in specific binding of insulin to male fetal liver membranes was exponential; in contrast, there was no apparent increase in specific binding to female fetal liver membranes during the gestational interval surveyed. Both the weights and the vertebral column lengths of these fetuses were shown by multivariate analysis to be significantly affected by the interaction between specific binding of insulin and fetal sex. However, in 30 additional sheep fetuses we observed no difference between male and female fetuses in the increase with time in liver glycogen content. The lack of sex difference in this postreceptor event is consonant with the demonstrated dissociation between liver insulin receptors and glycogen synthesis in the late fetal rat. Our observations suggest that late gestational differences between male and female sheep fetuses in insulin specific binding to liver and, possibly, to other tissues such as cartilage, muscle, and/or fat, that are coupled to postreceptor events may account for differences in fetal growth between the sexes.